Metabolic Inhibition Strongly Inhibits Na+-Dependent Mg2+ Efflux in Rat Ventricular Myocytes
Metabolic Inhibition Strongly Inhibits Na+-Dependent Mg2+ Efflux in Rat Ventricular Myocytes
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DOI：
10.1016/j.bpj.2009.02.013
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发表时间：
2009-06-17
影响因子：
3.4
通讯作者：
Konishi, Masato
中科院分区：
文献类型：
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作者：
Tashiro, Michiko;Inoue, Hana;Konishi, Masato
我们使用荧光指示剂furaptra（25℃）测量了大鼠心室肌细胞的细胞内Mg2+浓度（[Mg2+](i)）。在负载 Mg2+ 的正常供能细胞中，细胞外 Na+ 的引入导致 [Mg2+](i) 快速下降：[Mg2+](i) 下降的初始速率（初始 Delta[Mg2+](i)/Delta t）被认为代表了 Na+ 依赖性 Mg2+ 流出速率（假定的 Na+/Mg2+ 交换）。为了确定 Mg2+ 流出是否直接取决于细胞代谢产生的能量，除了跨膜 Na+ 梯度之外，我们还估计了代谢抑制后的初始 Delta[Mg2+](i)/Delta t。在缺乏细胞外 Na+ 和 Ca2+ 的情况下，用 1 μM 羰基氰化物对（三氟甲氧基）苯腙（一种线粒体解偶联剂）处理细胞，导致 [Mg2+](i) 在 5-8 分钟内从大约 0.9 mM 大幅增加到大约 2.5 mM（可能是因为 MgATP 的分解和 Mg2+ 的释放），并且细胞缩短至大约 50%。初始长度（可能是因为形成了严格的横桥）。应用 5 mM 氰化钾 (KCN)（呼吸抑制剂）>= 90 分钟后，观察到 [Mg2+](i) 和细胞缩短的类似增加。初始 Delta[Mg2+](i)/Delta t 在经羰基氰化物对（三氟甲氧基）苯腙处理的细胞中平均减少了 90%，在经 KCN 处理的细胞中平均减少了 92%。当用 5 mM KCN 处理细胞较短时间（59-85 分钟）时，观察到初始 Delta[Mg2+](i)/Delta t 显着下降（平均下降 59%），而细胞长度仅略有缩短。在初始 Delta[Mg2+](i)/Delta t 测量所需的时间内，用 Na+ 指示剂钠结合苯并呋喃间苯二甲酸酯估计的细胞内 Na+ 浓度 ([Na+](i)) 平均为 5.0-10.5 mM，远低于半抑制 Mg2+ 流出的 [Na+](i) 水平（类似于 40 mM）。使用 10 μM 尼日利亚菌素（一种 H+ 离子载体）使细胞内 pH 值正常化，并没有逆转 Mg2+ 流出的抑制。从这些结果来看，似乎是 ATP 降低到严格跨桥形成阈值以下（类似于本研究中间接估计的 0.4 mM），而不是 [Na+](i) 升高或细胞内酸中毒，抑制了 Mg2+ 流出，这表明 ATP 对于 Na+/Mg2+ 交换是绝对必要的。
We measured intracellular Mg2+ concentration ([Mg2+](i)) in rat ventricular myocytes using the fluorescent indicator furaptra (25 degrees C). In normally energized cells loaded with Mg2+, the introduction of extracellular Na+ induced a rapid decrease in [Mg2+](i): the initial rate of decrease in [Mg2+](i) (initial Delta[Mg2+](i)/Delta t) is thought to represent the rate of Na+-dependent Mg2+ efflux (putative Na+/Mg2+ exchange). To determine whether Mg2+ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na+ gradient, we estimated the initial Delta[Mg2+](i)/Delta t after metabolic inhibition. In the absence of extracellular Na+ and Ca2+, treatment of the cells with 1 mu M carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg2+](i) from similar to 0.9 mM to similar to 2.5 mM in a period of 5-8 min (probably because of breakdown of MgATP and release of Mg2+) and cell shortening to similar to 50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg2+](i) and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for >= 90 min. The initial Delta[Mg2+](i)/Delta t was diminished, on average, by 90% in carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone-treated cells and 92% in KCN-treated cells. When the cells were treated with 5 mM KCN for shorter times (59-85 min), a significant decrease in the initial Delta[Mg2+](i)/Delta t (on average by 59%) was observed with only a slight shortening of the cell length. Intracellular Na+ concentration ([Na+](i)) estimated with a Na+ indicator sodium-binding benzofuran isophthalate was, on average, 5.0-10.5 mM during the time required for the initial Delta[Mg2+](i)/Delta t measurements, which is well below the [Na+](i) level for half inhibition of the Mg2+ efflux (similar to 40 mM). Normalization of intracellular pH using 10 mu M nigericin, a H+ ionophore, did not reverse the inhibition of the Mg2+ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (similar to 0.4 mM estimated indirectly in the this study), rather than elevation of [Na+](i) or intracellular acidosis, inhibits the Mg2+ efflux, suggesting the absolute necessity of ATP for the Na+/Mg2+ exchange.